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The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.
Assuntos
Carcinogênese/patologia , Ciclo Celular , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Fígado/enzimologia , Fígado/patologia , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Idoso , Animais , Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Fígado/cirurgia , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 12 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Piridonas/farmacologia , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência , Especificidade por SubstratoRESUMO
BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. CONCLUSIONS: Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new in vitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.
RESUMO
BACKGROUND & AIMS: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood. METHODS: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. RESULTS: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. CONCLUSIONS: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. IMPACT AND IMPLICATIONS: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.
Assuntos
Reprogramação Celular , Hepatite Alcoólica , Animais , Camundongos , Hepatite Alcoólica/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Fígado/patologiaRESUMO
Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colangiocarcinoma/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Animais , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/fisiopatologia , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , PPAR alfa/genética , PPAR alfa/metabolismoRESUMO
Unfortunately, there is a gap of understanding in the pathophysiology of chronic liver disease due to the lack of experimental models that exactly mimic the human disease. Additionally, the diagnosis of patients is very poor due to the lack of biomarkers than can detect the disease in early stages. Thus, it is of utmost interest the generation of a multidisciplinary consortium from different countries with a direct translation. The present reports the meeting of the 2021 Iberoamerican Consortium for the study of liver Cirrhosis, held online, in October 2021. The meeting, was focused on the recent advancements in the field of chronic liver disease and cirrhosis with a specific focus on cell pathobiology and liver regeneration, molecular and cellular targets involved in non-alcoholic hepatic steatohepatitis, alcoholic liver disease (ALD), both ALD and western diet, and end-stage liver cirrhosis and hepatocellular carcinoma. In addition, the meeting highlighted recent advances in targeted novel technology (-omics) and opening therapeutic avenues in this field of research.
Assuntos
Hepatopatias Alcoólicas , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Cirrose Hepática/etiologia , Hepatopatias Alcoólicas/terapia , Hepatopatia Gordurosa não Alcoólica/terapia , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
OBJECTIVE: Despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). DESIGN: A prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. RESULTS: An initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. CONCLUSION: Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.
Assuntos
Neoplasias dos Ductos Biliares , Ácidos Nucleicos Livres , Colestase , Bile , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiopancreatografia Retrógrada Endoscópica , Colestase/etiologia , Colestase/genética , Constrição Patológica/diagnóstico , Detecção Precoce de Câncer , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.
Assuntos
Neoplasias dos Ductos Biliares , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma , DNA (Citosina-5-)-Metiltransferase 1 , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/fisiologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/efeitos dos fármacos , Código das Histonas/fisiologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Resultado do Tratamento , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).
Assuntos
Carcinoma Hepatocelular , Gastroenterologia , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/complicações , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/complicações , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/terapia , Hepatopatia Gordurosa não Alcoólica/patologia , Cirrose Hepática/complicações , Cirrose Hepática/patologiaRESUMO
Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Consenso , Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Europa (Continente) , Humanos , Fígado/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: An association between Crohn's disease (CD) and hepatic steatosis has been reported. However, the underlying mechanisms of steatosis progression in CD are not clear. Among the most effective CD treatments are agents that inhibit Tumor-Necrosis-Factor (TNF) activity, yet it is unclear why anti-TNFα agents would affect steatosis in CD. Recent studies suggest that microbiome can affect both, CD and steatosis pathogenesis. Therefore, we here analysed a potential relationship between anti-TNF treatment and hepatic steatosis in CD, focusing on the gut-liver axis. METHODS: This cross-sectional study evaluated patients with established CD, with and without anti-TNFα treatment, analysing serum markers of liver injury, measurement of transient elastography, controlled attenuation parameter (CAP) and MRI for fat detection. Changes in lipid and metabolic profiles were assessed by serum and stool lipidomics and metabolimics. Additionally, we analysed gut microbiota composition and mediators of bile acid (BA) signalling via stool and serum analysis. RESULTS: Patients on anti-TNFα treatment had less hepatic steatosis as assessed by CAP and MRI. Serum FGF19 levels were significantly higher in patients on anti-TNFα therapy and associate with reduced steatosis and increased bowel motility. Neutral lipids including triglycerides were reduced in the serum of patients on anti-TNF treatment. Bacteria involved in BA metabolism and FGF19 regulation, including Firmicutes, showed group-specific alterations with low levels in patients without anti-TNFα treatment. Low abundance of Firmicutes was associated with higher triglyceride levels. CONCLUSIONS: Anti-TNFα treatment is associated with reduced steatosis, lower triglyceride levels, alterations in FXR-signalling (eg FGF19) and microbiota composition in CD.
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Doença de Crohn , Fígado Gorduroso , Doença de Crohn/tratamento farmacológico , Estudos Transversais , Hormônios , Humanos , Inibidores do Fator de Necrose TumoralRESUMO
E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Ciclina E/biossíntese , Quinase 2 Dependente de Ciclina/biossíntese , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Proteínas Oncogênicas/biossíntese , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Ciclinas/biossíntese , Ciclinas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genéticaRESUMO
Animal models are crucial for improving our understanding of human pathogenesis, enabling researchers to identify therapeutic targets and test novel drugs. In the current review, we provide a comprehensive summary of the most widely used experimental models of chronic liver disease, starting from early stages of fatty liver disease (non-alcoholic and alcoholic) to steatohepatitis, advanced cirrhosis and end-stage primary liver cancer. We focus on aspects such as reproducibility and practicality, discussing the advantages and weaknesses of available models for researchers who are planning to perform animal studies in the near future. Additionally, we summarise current and prospective models based on human tissue bioengineering.
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Modelos Animais de Doenças , Hepatopatias , Animais , Descoberta de Drogas , Humanos , Hepatopatias/tratamento farmacológico , Hepatopatias/patologia , Hepatopatias/fisiopatologia , Reprodutibilidade dos Testes , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/tendênciasRESUMO
Ferroptosis has emerged as a new type of cell death in different pathological conditions, including neurological and kidney diseases and, especially, in different types of cancer. The hallmark of this regulated cell death is the presence of iron-driven lipid peroxidation; the activation of key genes related to this process such as glutathione peroxidase-4 (gpx4), acyl-CoA synthetase long-chain family member-4 (acsl4), carbonyl reductase [NADPH] 3 (cbr3), and prostaglandin peroxidase synthase-2 (ptgs2); and morphological changes including shrunken and electron-dense mitochondria. Iron overload in the liver has long been recognized as both a major trigger of liver damage in different diseases, and it is also associated with liver fibrosis. New evidence suggests that ferroptosis might be a novel type of non-apoptotic cell death in several liver diseases including non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), drug-induced liver injury (DILI), viral hepatitis, and hemochromatosis. The interaction between iron-related lipid peroxidation, cellular stress signals, and antioxidant systems plays a pivotal role in the development of this novel type of cell death. In addition, integrated responses from lipidic mediators together with free iron from iron-containing enzymes are essential to understanding this process. The presence of ferroptosis and the exact mechanisms leading to this non-apoptotic type of cell death in the liver remain scarcely elucidated. Recognizing ferroptosis as a novel type of cell death in the liver could lead to the understanding of the complex interaction between different types of cell death, their role in progression of liver fibrosis, the development of new biomarkers, as well as the use of modulators of ferroptosis, allowing improved theranostic approaches in the clinic.
Assuntos
Apoptose , Ferroptose , Fígado/patologia , Animais , Autofagia , Biomarcadores/metabolismo , Humanos , Ferro/metabolismoRESUMO
MicroRNA 155 (miR-155) is involved in immune and inflammatory diseases and is associated with liver fibrosis and steatohepatitis. However, the mechanisms involved in miR-155 regulation of liver injury are largely unknown. The role of miR-155 in acute liver injury was assessed in wild-type (WT), miR-155-/- , and miR-155-/- mice transplanted with WT bone marrow. Additionally, miR-155 expression was evaluated in liver tissue and peripheral blood mononuclear cells of patients with autoimmune hepatitis. Concanavalin A, but not acetaminophen, treatment increased the expression of miR-155 in liver tissue of WT mice. Concanavalin A induced increases in cell death, liver aminotransferases, and expression of proinflammatory cytokines (chemokine [C-X-C motif] ligands 1, 5, 9, 10, and 11; chemokine [C-C motif] ligands 2 and 20; and intercellular cell adhesion molecule 1) in miR-155-/- compared to WT mice. Importantly, these animals showed a significant decrease in cluster of differentiation 4-positive/chemokine (C-X-C motif) receptor 3-positive and forkhead box p3-positive cell recruitment but no changes in other inflammatory cell populations. Mechanistically, miR-155-deficient regulatory T cells showed increased SH2 domain-containing inositol 5-phosphatase 1 expression, a known target of miR-155. Inhibition of SH2 domain-containing inositol 5-phosphatase 1 in miR-155-/- mice restored forkhead box p3 recruitment and reduced liver cytokine expression. Transplantation of bone marrow from WT animals into miR-155-/- mice partially reversed the effect of concanavalin A on miR-155-/- mice as assessed by proinflammatory cytokines and cell death protein expression. Patients with autoimmune hepatitis showed a marked increase in miR-155 expression in the liver but reduced expression of miR-155 in peripheral blood mononuclear cells. CONCLUSION: miR-155 expression is altered in both liver tissue and circulating inflammatory cells during liver injury, thus regulating inflammatory cell recruitment and liver damage; these results suggest that maintaining miR-155 expression in inflammatory cells might be a potential strategy to modulate liver injury. (Hepatology 2018).
Assuntos
Hepatite Autoimune/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Concanavalina A/farmacologia , Citocinas/metabolismo , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/patologia , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
BACKGROUND & AIMS: Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. METHODS: Liver injury was characterized in a cohort of human sera (nâ¯=â¯28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3-/-) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28â¯days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. RESULTS: Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepamice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. CONCLUSION: These findings show that intervention against CASP8 in liver parenchymal cells - specifically in cholangiocytes - might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. LAY SUMMARY: Loss of caspase 8 - a protein involved in programmed cell death - in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.
Assuntos
Caspase 8/metabolismo , Colestase/patologia , Hepatócitos/metabolismo , Cirrose Hepática Biliar/patologia , Fígado/patologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Biópsia , Caspase 3/metabolismo , Caspase 8/genética , Inibidores de Caspase/farmacologia , Colestase/prevenção & controle , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Fibrose/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Necrose , Tecido Parenquimatoso/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Improvement in cognitive function after orthotopic liver transplantation (LT) has been demonstrated in the acute setting immediately after LT and in acute liver failure. However, the longterm changes in cerebral hemodynamics after LT remain unexplored. Therefore, we aimed to evaluate the longterm changes in cerebral hemodynamics of patients with cirrhosis after LT. In this prospective cohort study, we performed transcranial Doppler ultrasonography (TCD) measuring the pulsatility index (PI), resistance index (RI), and breath-holding index (BHI) to evaluate cerebrovascular structural integrity and reactivity, respectively, in both middle cerebral arteries before and after LT. Neuropsychometric tests and West-Haven criteria were used for hepatic encephalopathy (HE) characterization. Interleukin 6 and tumor necrosis factor α plasma levels were measured. Descriptive statistics and Wilcoxon's test were used. There were 27 patients who were included. Median follow-up after LT was 6 months, mean age before LT was 46.3 ± 10.3 years, the main etiology was hepatitis C virus (59%), and most of the patients were Child-Pugh B (15/27). Model for End-Stage Liver Disease (MELD) score was 16 ± 7.5, MELD-Na was 19.3 ± 7.1, Psychometric Hepatic Encephalopathy Score was -3.48 ± 3.66, and critical flicker fusion (CFF) was 40.28 ± 5.70 Hz. Before LT, 17/27 patients had HE and 11/27 ascites. A decrease of 20.8% and 13.5% in PI and RI was observed after LT (P < 0.001, both), together with an increase in BHI (32.4%, P = 0.122). These changes in cerebral hemodynamics paralleled those in systemic inflammation. Clinical improvement in cognition was observed in all patients with overt HE after LT. In conclusion, these results show a significant improvement in cerebral hemodynamics after LT, obtained through TCD, indicating less arterial cerebral vasoconstriction together with a decrease in systemic inflammation. Changes in cerebral vasoconstriction can be the basis for the improvement in cognitive function after LT in the long term.
Assuntos
Circulação Cerebrovascular/fisiologia , Hemodinâmica/fisiologia , Encefalopatia Hepática/diagnóstico , Cirrose Hepática/cirurgia , Transplante de Fígado , Adulto , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Cognição/fisiologia , Feminino , Seguimentos , Encefalopatia Hepática/etiologia , Encefalopatia Hepática/fisiopatologia , Humanos , Cirrose Hepática/complicações , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Artéria Cerebral Média/diagnóstico por imagem , Artéria Cerebral Média/fisiologia , Estudos Prospectivos , Psicometria , Índice de Gravidade de Doença , Resultado do Tratamento , Ultrassonografia Doppler TranscranianaRESUMO
Alcoholic liver disease (ALD) is the most common chronic liver disease in the Western world, and it persists at a high prevalence. Understanding the pathophysiology and successful treatment for ALD is closely associated with the suitability of the animal model, which fully reflects all aspects of the pathogenesis and typical histological findings. This study reviews one of the most widely used experimental models of ALD in rodents-the Lieber-DeCarli (LDC) liquid diet. It is an easy, accurate, reliable, and inexpensive model to study the pathogenesis of early stages of ALD. Here, we discuss the historical background and provide an overview of the advantages and disadvantages of the classical LDC as well as modified "second-hit" models. We also provide a comprehensive protocol for the application of the LDC diet to perform it successfully, reliably, and reproducibly in mice.